Amyloid State Research Articles

Clinical Features, Diagnosis and Treatment of amyloidosis: A Review

The name Amyloid is derived from the word "Oid", which means semi-starch, which is a protein that transforms into the shape or amyloid state when a distortion or a change occurs in its secondary structure as it becomes insoluble, shaped, and assembled as beta folded sheets. The structure of the amyloid protein can be seen by the electronic microscope as a group of non-branched fibers, with unspecified lengths and cross-sections between (7.5–10) nanometers. Also, the distinguished structure (the folded beta sheets) is clearly seen, and this structure is responsible for the unique pigmentation that is shown by the amyloid proteins with the Congo red stain.

Pregnenolone derivatives for the treatment of Alzheimer's disease: synthesis, and in vitro inhibition of amyloid β1-42 peptide aggregation, acetylcholinesterase and carbonic anhydrase-II.

The amyloid state, which is a specific conformation of proteins, offers valuable information about both functional protein structures and the pathological assemblies associated with various diseases. One of the major hallmarks of Alzheimer's disease includes primarily the extracellular build-up of a peptide known as amyloid-β, which has a sequence consisting of 39 to 42 amino acid residues, and the formation of intracellular neurofibrillary tangles mostly consisting of hyperphosphorylated tau protein. Drugs that are expected to reduce Aβ production, prevent Aβ aggregation, and promote Aβ clearance are promising approaches for treating AD. Current work is focused on identifying the compounds that have balanced even mild biological activities against multiple targets instead of finding one-target compound with high potency. We synthesized pregnenolone derivatives and evaluated their potential against inhibition of eeAChE/eqBChE, hCA-II and self-mediated Aβ1-42 peptide aggregation. Our synthesized derivatives 23, and 25-27 exhibited concomitant inhibition of all the tested macromolecular targets. All the active compounds were found to be BBB penetrants in the PAMPA assay. Furthermore, these selected compounds were found to be non-neurotoxic in the MTT assay on neuroblastoma SH-SY5Y cells. Docking studies support dual binding site (PAS and CAS) inhibition of AChE which showed Aβ1-42 aggregation and AChE inhibition. Moreover, docking studies carried out on the 3D crystallographic structure of Aβ1-42 peptide (PDB ID = 1IYT) showed significant interactions with amino acid residues Asp 23 and Lys 28, and hydrophobic interactions with the Phe19, Phe20, and Ala 30 effectively impeding the formation of β-sheet structures.

Non-fibril amyloid aggregation at the air/water interface: self-adaptive pathway resulting in a 2D Janus nanofilm.

The amyloid states of proteins are implicated in several neurodegenerative diseases and bioadhesion processes. However, the classical amyloid fibrillization mechanism fails to adequately explain the formation of polymorphic aggregates and their adhesion to various surfaces. Herein, we report a non-fibril amyloid aggregation pathway, with disulfide-bond-reduced lysozyme (R-Lyz) as a model protein under quasi-physiological conditions. Very different from classical fibrillization, this pathway begins with the air-water interface (AWI) accelerated oligomerization of unfolded full-length protein, resulting in unique plate-like oligomers with self-adaptive ability, which can adjust their conformations to match various interfaces such as the asymmetric AWI and amyloid-protein film surface. The pathway enables a stepwise packing of the plate-like oligomers into a 2D Janus nanofilm, exhibiting a divergent distribution of hydrophilic/hydrophobic residues on opposite sides of the nanofilm. The resulting Janus nanofilm possesses a top-level Young's modulus (8.3 ± 0.6 GPa) among amyloid-based materials and exhibits adhesive strength two times higher (145 ± 81 kPa) than that of barnacle cement. Furthermore, we found that such an interface-directed pathway exists in several amyloidogenic proteins with a similar self-adaptive 2D-aggregation process, including bovine serum albumin, insulin, fibrinogen, hemoglobin, lactoferrin, and ovalbumin. Thus, our findings on the non-fibril self-adaptive mechanism for amyloid aggregation may shed light on polymorphic amyloid assembly and their adhesions through an alternative pathway.

Phase separation modulates the functional amyloid assembly of human CPEB3

How functional amyloids are regulated to restrict their activity is poorly understood. The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that adopts an amyloid state key for memory persistence. Its monomer represses the translation of synaptic target mRNAs while phase separated, whereas its aggregated state acts as a translational activator. Here, we have explored the sequence-driven molecular determinants behind the functional aggregation of human CPEB3 (hCPEB3). We found that the intrinsically disordered region (IDR) of hCPEB3 encodes both an amyloidogenic and a phase separation domain, separated by a poly-A-rich region. The hCPEB3 amyloid core is composed by a hydrophobic region instead of the Q-rich stretch found in the Drosophila orthologue. The hCPEB3 phase separation domain relies on hydrophobic interactions with ionic strength dependence, and its droplet ageing process leads to a liquid-to-solid transition with the formation of a non-fibril-based hydrogel surrounded by starburst droplets. Furthermore, we demonstrate the differential behavior of the protein depending on its environment. Under physiological-like conditions, hCPEB3 can establish additional electrostatic interactions with ions, increasing the stability of its liquid droplets and driving a condensation-based amyloid pathway.

OmpC and OmpF Outer Membrane Proteins of Escherichia coli and Salmonella enterica Form Bona Fide Amyloids.

Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the β-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their β-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-β that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli, and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis.

A Hairpin Motif in the Amyloid-β Peptide Is Important for Formation of Disease-Related Oligomers.

The amyloid-β (Aβ) peptide is associated with the development of Alzheimer's disease and is known to form highly neurotoxic prefibrillar oligomeric aggregates, which are difficult to study due to their transient, low-abundance, and heterogeneous nature. To obtain high-resolution information about oligomer structure and dynamics as well as relative populations of assembly states, we here employ a combination of native ion mobility mass spectrometry and molecular dynamics simulations. We find that the formation of Aβ oligomers is dependent on the presence of a specific β-hairpin motif in the peptide sequence. Oligomers initially grow spherically but start to form extended linear aggregates at oligomeric states larger than those of the tetramer. The population of the extended oligomers could be notably increased by introducing an intramolecular disulfide bond, which prearranges the peptide in the hairpin conformation, thereby promoting oligomeric structures but preventing conversion into mature fibrils. Conversely, truncating one of the β-strand-forming segments of Aβ decreased the hairpin propensity of the peptide and thus decreased the oligomer population, removed the formation of extended oligomers entirely, and decreased the aggregation propensity of the peptide. We thus propose that the observed extended oligomer state is related to the formation of an antiparallel sheet state, which then nucleates into the amyloid state. These studies provide increased mechanistic understanding of the earliest steps in Aβ aggregation and suggest that inhibition of Aβ folding into the hairpin conformation could be a viable strategy for reducing the amount of toxic oligomers.

Alpha-synuclein and the Parkinson's disease drug pipeline

The process of protein aggregation involves the transformation of soluble peptides into insoluble cross-beta amyloids. In Parkinson's disease (PD), soluble monomeric α-synuclein transforms into the amyloid state known as Lewy pathology. Monomeric (functional) α-synuclein depletes as the fraction of Lewy pathology increases. We examined the allocation of disease-modifying projects in the PD therapeutic pipeline classified based on whether they aimed to reduce directly or indirectly the insoluble or increase the soluble α-synuclein. A project was defined as a drug development program that may include more than one registered clinical trial, according to the Parkinson's Hope List, a database of therapies under development for PD. Of 67 projects, 46 aimed to reduce α-synuclein, 15 (22.4%) directly and 31 (46.3%) indirectly, amounting to 68.7% of all disease-modifying projects. No projects explicitly aimed to increase soluble α-synuclein levels. Altogether, α-synuclein is the target of more than two-thirds of the disease-modifying pipeline, with treatments aimed at reducing or preventing an increase in its insoluble fraction. As no treatments aim to restore soluble α-synuclein levels within a normal range, we propose rebalancing the therapeutic PD pipeline.

PRIMA-1 inhibits Y220C p53 amyloid aggregation and synergizes with cisplatin in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Although many therapeutic options are available, several factors, including the presence of p53 mutations, impact tumor development and therapeutic resistance. TP53 is the second most frequently mutated gene in HCC, comprising more than 30% of cases. Mutations in p53 result in the formation of amyloid aggregates that promote tumor progression. The use of PRIMA-1, a small molecule capable of restoring p53, is a therapeutic strategy to pharmacologically target the amyloid state mutant p53. In this study, we characterize an HCC mutant p53 model for the study of p53 amyloid aggregation in HCC cell lines, from in silico analysis of p53 mutants to a 3D-cell culture model and demonstrate the unprecedented inhibition of Y220C mutant p53 aggregation by PRIMA-1. In addition, our data show beneficial effects of PRIMA-1 in several "gain of function" properties of mutant-p53 cancer cells, including migration, adhesion, proliferation, and drug resistance. We also demonstrate that the combination of PRIMA-1 and cisplatin is a promising approach for HCC therapy. Taken together, our data support the premise that targeting the amyloid-state of mutant p53 may be an attractive therapeutic approach for HCC, and highlight PRIMA-1 as a new candidate for combination therapy with cisplatin.

Abstract 4820: The disassembly of amyloid-bodies

Abstract The ability of cells to adapt to a wide variety of stress conditions plays a critical role in various physiological and pathological settings, including development, cancer and neurological disorders. We recently reported the discovery of stress-induced low complexity noncoding RNA derived from stimuli-specific loci of the ribosomal intergenic spacer (rIGSRNA); an enigmatic region of the human genome historically dismissed as “junk” DNA. We showed that low complexity rIGSRNA activate a physiological amyloidogenic program that converts the nucleolus into Amyloid-bodies: a molecular prison of immobilized proteins in an amyloid-like state. This conserved post-translational regulatory pathway enables cells to rapidly and reversibly store an array of endogenous proteins in Amyloid-bodies and enter a dormant-like phenotype in response to severe environmental insults. While many membrane-less compartments have been described as liquid-like (e.g., stress granules, P-bodies, germ cell granules), our discovery of Amyloid-bodies provided evidence of an amyloidogenic process that can physiologically transition biological matter to a solid-like state. The ability of mammalian cells to efficiently dissemble Amyloid-bodies raises a fundamental question: Are mammalian cell disaggregases involved in Amyloid-body disassembly? Here, we show that Amyloid-bodies undergo a solid-to-liquid-phase transition to release sequestered proteins and restore nucleolar functions. An RNAi screened identified key Heat Shock Proteins (Hsps) that remodel Amyloid-bodies back to the liquid nucleoli on stress termination. The composition of mammalian disaggregases differs considerably from non-metazoans and those involved in disassembly of pathological amyloids in vitro. Activation of mammalian disaggregases and Amyloid-body disassembly is dependent on ATP concentration. Conceptually, this work identifies metazoan disaggregases challenging the widely accepted paradigm that the amyloid state is irreversible in mammalian cells. The data also provides alternative insights into pathogenic amyloids by examining their disassembly in cellular systems. Citation Format: Chloe Kirk, Michael Bokros, Alex Grunfeld, Stephen Lee. The disassembly of amyloid-bodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4820.

Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains.

Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.

Sequence-Based Prediction of Protein Phase Separation: The Role of Beta-Pairing Propensity.

The formation of droplets of bio-molecular condensates through liquid-liquid phase separation (LLPS) of their component proteins is a key factor in the maintenance of cellular homeostasis. Different protein properties were shown to be important in LLPS onset, making it possible to develop predictors, which try to discriminate a positive set of proteins involved in LLPS against a negative set of proteins not involved in LLPS. On the other hand, the redundancy and multivalency of the interactions driving LLPS led to the suggestion that the large conformational entropy associated with non specific side-chain interactions is also a key factor in LLPS. In this work we build a LLPS predictor which combines the ability to form pi-pi interactions, with an unrelated feature, the propensity to stabilize the β-pairing interaction mode. The cross-β structure is formed in the amyloid aggregates, which are involved in degenerative diseases and may be the final thermodynamically stable state of protein condensates. Our results show that the combination of pi-pi and β-pairing propensity yields an improved performance. They also suggest that protein sequences are more likely to be involved in phase separation if the main chain conformational entropy of the β-pairing maintained droplet state is increased. This would stabilize the droplet state against the more ordered amyloid state. Interestingly, the entropic stabilization of the droplet state appears to proceed according to different mechanisms, depending on the fraction of "droplet-driving" proteins present in the positive set.

Sequence-based Prediction of the Cellular Toxicity Associated with Amyloid Aggregation within Protein Condensates.

Various neurological dysfunctions are associated with cytotoxic amyloid-containing aggregates formed through the irreversible maturation of protein condensates generated by phase separation. Here, we investigate the amino acid code for this cytotoxicity using TDP-43 deep-sequencing data. Within the droplet landscape framework, we analyze the impact of mutations in the amyloid core, aggregation hot-spot, and droplet-promoting residues on TDP-43 cytotoxicity. Our analysis suggests that TDP-43 mutations associated with low cytotoxicity moderately decrease the probability of droplet formation while increasing the probability of multimodal binding. These mutations promote both ordered and disordered binding modes, thus facilitating the conversion between the droplet and amyloid states. Based on this understanding, we develop an extension of the FuzDrop method for the sequence-based prediction of the cytotoxicity of aging condensates and test it over 20,000 TDP-43 variants. Our analysis provides insight into the amino acid code that regulates the cytotoxicity associated with the maturation of liquid-like condensates into amyloid-containing aggregates, suggesting that, at least in the case of TDP-43, mutations that promote aggregation tend to decrease cytotoxicity, while those that promote droplet formation tend to increase cytotoxicity.

RopB protein of Rhizobium leguminosarum bv. viciae adopts amyloid state during symbiotic interactions with pea (Pisum sativum L.).

Amyloids represent protein aggregates with highly ordered fibrillar structure associated with the development of various disorders in humans and animals and involved in implementation of different vital functions in all three domains of life. In prokaryotes, amyloids perform a wide repertoire of functions mostly attributed to their interactions with other organisms including interspecies interactions within bacterial communities and host-pathogen interactions. Recently, we demonstrated that free-living cells of Rhizobium leguminosarum, a nitrogen-fixing symbiont of legumes, produce RopA and RopB which form amyloid fibrils at cell surface during the stationary growth phase thus connecting amyloid formation and host-symbiont interactions. Here we focused on a more detailed analysis of the RopB amyloid state in vitro and in vivo, during the symbiotic interaction between R. leguminosarum bv. viciae with its macrosymbiont, garden pea (Pisum sativum L.). We confirmed that RopB is the bona fide amyloid protein since its fibrils exhibit circular x-ray reflections indicating its cross-β structure specific for amyloids. We found that fibrils containing RopB and exhibiting amyloid properties are formed in vivo at the surface of bacteroids of R. leguminosarum extracted from pea nodules. Moreover, using pea sym31 mutant we demonstrated that formation of extracellular RopB amyloid state occurs at different stages of bacteroid development but is enhanced in juvenile symbiosomes. Proteomic screening of potentially amyloidogenic proteins in the nodules revealed the presence of detergent-resistant aggregates of different plant and bacterial proteins including pea amyloid vicilin. We demonstrated that preformed vicilin amyloids can cross-seed RopB amyloid formation suggesting for probable interaction between bacterial and plant amyloidogenic proteins in the nodules. Taken together, we demonstrate that R. leguminosarum bacteroids produce extracellular RopB amyloids in pea nodules in vivo and these nodules also contain aggregates of pea vicilin amyloid protein, which is able to cross-seed RopB fibrillogenesis in vitro. Thus, we hypothesize that plant nodules contain a complex amyloid network consisting of plant and bacterial amyloids and probably modulating host-symbiont interactions.

Succinct Amyloid and Nonamyloid Patterns in Hexapeptides

Hexapeptides are widely applied as a model system for studying the amyloid-forming properties of polypeptides, including proteins. Recently, large experimental databases have become publicly available with amyloidogenic labels. Using these data sets for training and testing purposes, one may build artificial intelligence (AI)-based classifiers for predicting the amyloid state of peptides. In our previous work (Biomolecules2021, 11, 500), we described the Support Vector Machine (SVM)-based Budapest Amyloid Predictor (https://pitgroup.org/bap). Here, we apply the Budapest Amyloid Predictor for discovering numerous amyloidogenic and nonamyloidogenic hexapeptide patterns with accuracy between 80% and 84%, as surprising and succinct novel rules for further understanding the amyloid state of peptides. For example, we have shown that for any independently mutated residue (position marked by “x”), the patterns CxFLWx, FxFLFx, or xxIVIV are predicted to be amyloidogenic, while those of PxDxxx, xxKxEx, and xxPQxx are nonamyloidogenic. We note that each amyloidogenic pattern with two x’s (e.g.,CxFLWx) describes succinctly 202 = 400 hexapeptides, while the nonamyloidogenic patterns comprising four point mutations (e.g.,PxDxxx) give 204 = 160 000 hexapeptides in total. We also examine the restricted substitutions for positions “x” from subclasses of proteinogenic amino acid residues; for example, if “x” is substituted with hydrophobic amino acids, then there exist patterns containing three x’s, like MxVVxx, predicted to be amyloidogenic. If we can choose for the x positions any hydrophobic amino acids, except the “structure breaker” proline, then we get amyloid patterns with five x positions, for example, xxxFxx, each corresponding to 32 768 hexapeptides. To our knowledge, no similar applications of artificial intelligence tools or succinct amyloid patterns were described before the present work.

Investigating the Sequence Determinants of the Curling of Amyloid Fibrils Using Ovalbumin as a Case Study.

Highly ordered, straight amyloid fibrils readily lend themselves to structure determination techniques and have therefore been extensively characterized. However, the less ordered curly fibrils remain relatively understudied, and the structural organization underlying their specific characteristics remains poorly understood. We found that the exemplary curly fibril-forming protein ovalbumin contains multiple aggregation prone regions (APRs) that form straight fibrils when isolated as peptides or when excised from the full-length protein through hydrolysis. In the context of the intact full-length protein, however, the regions separating the APRs facilitate curly fibril formation. In fact, a meta-analysis of previously reported curly fibril-forming proteins shows that their inter-APRs are significantly longer and more hydrophobic when compared to straight fibril-forming proteins, suggesting that they may cause strain in the amyloid state. Hence, inter-APRs driving curly fibril formation may not only apply to our model protein but rather constitute a more general mechanism.

Interaction of Novel Monomethine Cyanine Dyes with Proteins in Native and Amyloid States

Molecular interactions between novel monomethine cyanine dyes and non-fibrilar and fibrilar proteins were assessed using fluorescence spectroscopy and molecular docking techniques. To this end, the fluorescence spectral properties of dyes have been explored in the buffer solution and in the presence of insulin and lysozyme in the native and amyloid states. It was observed that association of monomethines with the native and fibrillar proteins was accompanied with a significant enhancement of the fluorophore fluorescence, being more pronounced in the presence of aggregated insulin and lysozyme. The quantitative information about the dye-protein binding was obtained through approximating the experimental dependencies of the fluorescence intensity increase vs protein concentration by the Langmuir model. Analysis of the spectral properties and the binding characteristics of monomethines in the presence of the fibrillar insulin and lysozyme showed that the introduction of chloro- and fluorine-substitutients to the oxazole yellow derivatives, as well as the long aliphatic substitution on the nitrogen atom of the benzazole chromophore of YO-dyes had a negative impact on the dye amyloid specificity. Molecular docking studies showed that monomethines tend to form the most stable complexes with the B-chain residues Val 17, Leu17, Ala 14, Phe1, Gln 4 and Leu 6 and the A-chain residue Leu 13, Tyr 14, Glu 17 of non-fibrilar insulin and interact with the deep cleft of native lysozyme lined with both hydrophobic (Ile98, Ile 58, Thr108, Thr 62 and Thr 63 residues) and negatively (Asp101, Asp 107) charged residues. The wet surface groove Gln15_Glu17 and groove G2-L4/S8-W10 were found as the most energetically favorable binding sites for examined monomethine dyes in the presence of the insulin and lysozyme fibrils, respectively.

FuzDrop on AlphaFold: visualizing the sequence-dependent propensity of liquid-liquid phase separation and aggregation of proteins.

Many proteins perform their functions within membraneless organelles, where they form a liquid-like condensed state, also known as droplet state. The FuzDrop method predicts the probability of spontaneous liquid–liquid phase separation of proteins and provides a sequence-based score to identify the regions that promote this process. Furthermore, the FuzDrop method estimates the propensity of conversion of proteins to the amyloid state, and identifies aggregation hot-spots, which can drive the irreversible maturation of the liquid-like droplet state. These predictions can also identify mutations that can induce formation of amyloid aggregates, including those implicated in human diseases. To facilitate the interpretation of the predictions, the droplet-promoting and aggregation-promoting regions can be visualized on protein structures generated by AlphaFold. The FuzDrop server (https://fuzdrop.bio.unipd.it) thus offers insights into the complex behavior of proteins in their condensed states and facilitates the understanding of the functional relationships of proteins.

The Role of Heat Shock Proteins in Amyloid Body Reversal

The ability of cells to adapt to a wide variety of stress conditions plays a critical role in various physiological and pathological settings, including development, cancer and neurological disorders. We recently reported the surprising discovery of stress‐induced low complexity noncoding RNA derived from stimuli‐specific loci of the ribosomal intergenic spacer (rIGSRNA); an enigmatic region of the human genome historically dismissed as “junk” DNA. We showed that low complexity rIGSRNA activate a physiological amyloidogenic program that converts the nucleolus into Amyloid Bodies: a molecular prison of immobilized proteins in an amyloid‐like state. This conserved post‐translational regulatory pathway enables cells to rapidly and reversibly store an array of endogenous proteins in Amyloid‐bodies and enter quiescence in response to severe environmental insults. While many membrane‐less compartments have been described as liquid‐like (e.g., stress granules, P‐bodies, germ cell granules), our discovery of Amyloid‐bodies provided evidence of an amyloidogenic process that can physiologically transition biological matter to a solid‐like state. The ability of mammalian cells to efficiently dissemble Amyloid‐bodies raises a fundamental question: Are mammalian cell disaggregases involved in Amyloid‐body disassembly? Here, we show that Amyloid‐bodies undergo a multi‐step process solid‐to‐liquid‐phase transition to release sequestered proteins and restore nucleolar functions. An RNAi screened identified key heat shock proteins (hsps) that remodel Amyloid‐bodies back to the liquid nucleoli on stress termination. The composition of mammalian disaggregases differs considerably from non‐metazoans and those involved in disassembly of pathological amyloids in vitro. Activation of mammalian disaggregases and Amyloid‐body disassembly is dependent on ATP concentration. Conceptually, this work identifies metazoan disaggregases challenging the widely accepted paradigm that the amyloid state is irreversible in mammalian cells. The data also provides alternative insights into pathogenic amyloids by examining their disassembly in cellular systems.

OmpF porins of Enterobacteriaceae possess amyloidogenic properties

Outer membrane porins represent an important group of bacterial virulence associated proteins, whose amyloidogenic properties are actively investigated. Amyloids are protein fibrils with highly ordered beta‐sheet‐rich spatial structure and unique physicochemical properties including unusual resistance to treatment with different detergents and proteases. In prokaryotes, at least ten different functional groups of functional amyloids have been identified. Several outer membrane porins of gram‐negative bacteria were shown to adopt amyloid state. In this study, we analyzed amyloid properties of the OmpF porins of Escherichia coli and Salmonella enteritidis species which are known to be associated with the virulence and pathogenic properties of these bacteria in humans and animals. Our data show that recombinant OmpF proteins form fibrils with morphology typical for amyloids. The in vitro obtained aggregates of OmpF demonstrate resistance to treatment with cold ionic detergents and proteases. Moreover, these aggregates bind amyloid‐specific dye thioflavin T and exhibit apple‐green birefringence in polarized light upon staining with Congo Red dye. Overall, these data confirm the amyloidogenic properties of the OmpF porins of E. coli and S. enteritidis in vitro and suggest that these proteins could represent novel functional bacterial amyloids probably associated with pathogen‐host interactions.

Proteins Do Not Replicate, They Precipitate: Phase Transition and Loss of Function Toxicity in Amyloid Pathologies.

Simple SummaryAmyloid aggregation of proteins in disease has been known for over a hundred years; however, effective therapeutics for amyloid pathologies such as Alzheimer’s disease and Parkinson’s disease are still lacking. This review divides the amyloid phenomenon into four major questions: What are amyloids? How do amyloids form? Can proteins replicate? How do amyloids cause toxicity? The aim is to answer these questions within a unified physicochemical framework that links the structural biology of amyloids to the thermodynamics of amyloid formation and the pathophysiology of amyloid aggregates in different diseases. We illustrate that the thermodynamics of protein aggregation does not support the prion protein-only replication hypothesis, and how the structural biology of amyloids makes them largely domainless, generic, and inert. The implications of this understanding for the etiology, pathogenesis and potential therapeutics of amyloid diseases are briefly discussed.Protein aggregation into amyloid fibrils affects many proteins in a variety of diseases, including neurodegenerative disorders, diabetes, and cancer. Physicochemically, amyloid formation is a phase transition process, where soluble proteins are transformed into solid fibrils with the characteristic cross-β conformation responsible for their fibrillar morphology. This phase transition proceeds via an initial, rate-limiting nucleation step followed by rapid growth. Several well-defined nucleation pathways exist, including homogenous nucleation (HON), which proceeds spontaneously; heterogeneous nucleation (HEN), which is catalyzed by surfaces; and seeding via preformed nuclei. It has been hypothesized that amyloid aggregation represents a protein-only (nucleic-acid free) replication mechanism that involves transmission of structural information via conformational templating (the prion hypothesis). While the prion hypothesis still lacks mechanistic support, it is also incompatible with the fact that proteins can be induced to form amyloids in the absence of a proteinaceous species acting as a conformational template as in the case of HEN, which can be induced by lipid membranes (including viral envelopes) or polysaccharides. Additionally, while amyloids can be formed from any protein sequence and via different nucleation pathways, they invariably adopt the universal cross-β conformation; suggesting that such conformational change is a spontaneous folding event that is thermodynamically favorable under the conditions of supersaturation and phase transition and not a templated replication process. Finally, as the high stability of amyloids renders them relatively inert, toxicity in some amyloid pathologies might be more dependent on the loss of function from protein sequestration in the amyloid state rather than direct toxicity from the amyloid plaques themselves.